Fermented dairy products are normally prepared by culturing compositions with appropriate microorganisms to yield products such as yogurts, buttermilks, and sour creams. For example, yogurt is generally made by fermenting milk with a culture that contains thermophilic organisms such as Streptococcus salivarius subsp. thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB). Additional cultures such as Lactobacillus acidophilus and bifidobacteria may also be included. Thermophilic cultures have an optimum growth temperature around 40.degree. C. It is known that acid production in yogurt is first initiated by ST followed by LB (Pette et al., 1950, Neth. Milk Dairy Journal 4:197-208). It is an intrinsic attribute of ST and LB that they do not grow at 15.degree. C. or lower (Rasic et al., Yogurt, 1978, Page 227). Nevertheless the production of acidic fermentation products in these organisms can continue during storage without accompanying growth. The carbon source for such acidic fermentation products includes saccharides present in the dairy products used in the fermentation, such as lactose, for example. The production of acidic products by ST generally stops at pH values of about 3.9-4.3 and that by LB at pH values of about 3.5-3.8 (Rasic et al.). This continued production of acidic products in the absence of growth of the culture is termed uncoupled acid production. Due to the strong tendency for uncoupled acid production, yogurt found in the marketplace often suffers from the accumulation of excess acidic products which occurs during the storage involved in distribution and sale. Excess acidic products are often accompanied by development of bitterness.
Nisin is a peptide-like antibacterial substance produced by Lactococcus lactis subsp. lactis (formerly known as Streptococcus lactis). Its structure is illustrated in U.S. Pat. No. 5,527,505 to Yamauchi et al. The highest activity preparations contain about 40 million IU per gram. A commercial preparation, NISAPLIN.RTM., containing about 1 million IU per gram is available from Aplin & Barrett Ltd., Beaminster, Dorset, England. Nisin has no known toxic effects in humans, and is widely used in a variety of prepared dairy foods.
A number of efforts have been reported since 1975 directed to reducing uncoupled acid production by controlling the post-fermentation acidification of yogurt. In some of these studies, a nisin producing culture was introduced in an attempt to inhibit these effects. Kalra et al. (Indian Journal of Dairy Science 28: 71-72 (1975)) incorporated the nisin producing culture Streptococcus lactis (now known as L. lactis subsp. lactis) along with the yogurt culture before fermentation. Others introduced nisin in milk prior to fermentation (Bayoumi, Chem. mikrobiol. technol. lebensm. 13:65-69 (1991)) or following fermentation (Gupta et al., Cultured Dairy Products Journal 23: 17-18 (1988); Gupta et al., Cultured Dairy Products Journal 23: 9-10 (1989)). In all cases, the rate of post-fermentation acidification was only partially inhibited by these treatments and the yogurt continued to become more acidic throughout its shelf life. To the extent that saccharides such as lactose contribute to the production of excess acidic products, attempts have been made to minimize this phenomenon by removing the saccharides prior to fermentation, by procedures such as ultrafilitration and diafiltration, for example.
In U.S. Pat. No. 5,527,505, by Yamauchi et al., yogurt from raw milk was produced by incorporating a nisin producing strain, Lactococcus lactis subsp. lactis, along with the traditional yogurt culture consisting of ST and LB. Yamauchi et al. teach that the lactococci are needed to secrete the nisin, whose effect is to retard the activity of ST and LB. The resulting yogurt therefore contains the lactococci used to produce the nisin. Nonetheless, the acidity of yogurt containing the nisin-producing bacteria increased by 64% to 96% in 14 days, in various experiments inoculated with differing amounts of L. lactis subsp. lactis, compared to the initial acidity at the completion of fermentation. Other studies (Hogarty et al., J. Fd. Prot. 45:1208-1211 (1982); Sadovski et al., XX International Dairy Congress, Vol. E: 542-5-44 (1978)) also noted acid production and development of bitterness at low temperature by some mesophilic starter lactococci in dairy products.
Chung et al. (Appl. Envir. Microbiol. 55, 1329-1333 (1989)) report that nisin has an inhibitory effect on gram-positive bacteria, such as L. monocytogenes, Staphylococcus aureus and Streptococcus lactis, but has no such effect on gram-negative bacteria such as Serratia marcescens, Salmonella typhimurium and Pseudomonas aeruginosa when these microorganisms are attached to meat.
In U.S. Pat. No. 5,015,487 to Collison et al., the use of nisin, as a representative of the class of lanthionine bacteriocins, to control undesirable microorganisms in heat processed meats is disclosed. In tests involving dipping frankfurters in nisin solutions, the growth of L. monocytogenes was effectively inhibited upon storage at 4.degree. C.
Nisin has been added to cheeses to inhibit toxin production by Clostridium botulinum (U.S. Pat. No. 4,584,199 to Taylor). U.S. Pat. No. 4,597,972 to Taylor discloses a detailed example in which chicken frankfurter components are shown to require the presence of both added nitrite and added nisin in order to prevent or delay botulinum toxin production when challenged with C. botulinum.
Maas et al. (Appl. Envir. Microbiol. 55, 2226-2229 (1989)) report that lactate, when incorporated into a turkey meat vacuum-packed composition, delays the generation of botulinum toxin in a manner directly dependent on the concentration of lactate introduced into the composition. Maas et al. do not mention nisin.
In U.S. Pat. Nos. 4,888,191 and 5,017,391, Anders et al. disclose compositions and methods related to the use of lactate salts to delay C. botulinum growth in a foodstuff such as fish or poultry. The foods are heated to a temperature sufficient to cook the meat but not to sterilize the product. Anders et al. suggest that lactate may be used alone, or in combination with other agents such as sodium nitrite. These patents fail to discuss nisin or its properties.
For chocolate-flavored yogurt, an increase in yogurt acidity during storage is especially troublesome because cocoa flavor tends to become more bitter with increasing acidity (The Wall Street Journal A13A, Jun. 20, 1995). There is also a loss of the desired dark, rich color of the chocolate with increasing acidity. Generally, for chocolate yogurt the pH of yogurt fermentation should not fall below about 5.5 throughout its refrigerated shelf life (generally about eight weeks) in order that the product retains a hint of yogurt flavor with an indulgent flavor of chocolate.
There currently does not appear to be any method that can minimize or eliminate the acidity increase in yogurt during storage, nor a method to minimize or prevent the fermentation of saccharides such as lactose and/or galactose that may be present. Attempts to arrest the production of acid by yogurt cultures, by the addition of nisin before or after yogurt fermentation or the addition of a nisin producing culture to a yogurt culture before or after milk fermentation, have not been successful. The need to arrest uncoupled acid production, and to minimize the fermentation of saccharides thus remains. This unsatisfied need extends as well to the lack of a method to inhibit the concomitant production of bitterness accompanying the uncoupled acid production. Finally for flavored fermented dairy products, this need is of special significance since excess acidity diminishes the flavor and stability of added flavorings such as chocolate.